The genome is the blueprint of an organism, and in human, it is made up of about three billion pairs of deoxyribonucleotides (DNA). In the genomic sequence, multiple sites differing among individuals of the same species have been discovered, and these are called polymorphism. In particular, the polymorphism due to a single nucleotide substitution is called single nucleotide polymorphism (SNP). Variations in SNP may sometimes have influence on diseases and drug efficacy. Thus, SNP is paid attention to as a possible factor to explain differences among individuals.
From the genome, mRNA, rRNA, and tRNA are expressed, and specific proteins and the like are selectively synthesized from these blueprints, supporting life activities. Studies on the presence or absence of expression of these RNAs and on their sequences are also important to elucidate life phenomena. The sequences of rRNAs are partially different depending on species, and investigation of their sequences makes it possible to identify species, and so forth.
For these purposes, it is essential to amplify the target sequence as well as to detect the amplified product by labeling in order to acquire recognizable information. A general method for amplifying a nucleic acid sequence known at present is polymerase chain reaction (PCR). PCR represents an amplification reaction in which a pair of primers is designed to sandwich a region desired to be amplified from a template DNA sequence; a reaction solution containing these primers, dNTP serving as substrates, a thermostable DNA polymerase, and the like is prepared; and a reaction cycle of heat denaturation, annealing, and extension, carried out at different temperatures, is repeated 20 to 30 times to amplify the region.
As the method for labeling amplified products, there is a visualization method, after amplification, in which the presence or absence of amplification by PCR is examined by electrophoresis in agarose gel and subsequent dyeing of double-stranded DNA with ethidium bromide and the like. Further, detection using the principle of specific binding to complementary chain necessitates direct labeling such as incorporation of a fluorescent substance into amplified products. The direct method for labeling amplification products includes a method in which the amplification reaction is carried out by binding in advance a fluorescent label, biotin, or the like to a primer, and a method in which a substrate containing a radioisotope, labeled with a fluorophore, or bound with biotin is allowed to be incorporated into amplified products during amplification reaction.
Although PCR in principle is able to amplify a very minute quantity of a template DNA sequence to a large quantity, there are certain cases in practice where amplification reaction does not take place or an incorrect region is amplified. Particularly when a partial region of a genome is amplified, the quantity of the template DNA to be amplified is extremely small relative to the quantity of the total DNA. Owing to non-specific binding of primers under the circumstances, it frequently occurs that amplification reaction does not take place or an incorrect region is amplified, thus making it difficult to obtain the target amplification products.
The amplified DNA must be labeled in a certain way in order to acquire objective information. Since any method of labeling after PCR requires much expense in time and effort, expensive reagents such as enzyme, and the like, it is costly compared with a method of labeling during PCR amplification reaction. Particularly when a large number of samples must be processed, this cost gives rise to a problem. However, it is not actually easy to perform labeling during PCR amplification reaction as well as acquiring the amplified products from a genome or the like at the same time.
When the presence or absence of binding between labeled amplification products and a probe prepared to be complementary to the former to hybridize in a sequence-specific manner is detected, the amplification products do not continue to bind to the probe but eventually form stable double strands with their complementary chains of the amplified products because the amplification products are double-stranded by nature and present in excess in terms of the number of molecules. In other words, the detection sensitivity becomes very low when only a labeling substance is simply incorporated into the amplification products.
In an attempt to solve the problem by allowing amplified products to be biased toward formation of single-stranded DNA, an asymmetrical PCR method has been devised (reference; “PCR Method of Gene Amplification: Basics and New Developments”, By Ikunosin Kato, Ed. Fujinaga, Kyoritsu Shuppan Co. Ltd., pp. 7–26 (Dec. 10, 1990)). In this method, a pair of primers for use in amplification reaction is supplied in unequal quantities rather than equal quantities. However, it is practically not easy to obtain amplification products stably from a genome and the like by the asymmetrical PCR method. Further, when the primers are present in excess even though present in unequal proportions, amplification reaction similar to an ordinary PCR occurs, and thus the problem was not solved.